1. Field of the Invention
The invention in the field of cell biology, physiology and medicine relates to a purified mammalian adipocyte polypeptide, DNA encoding the polypeptide, antibodies thereto, and methods to determine susceptibility to obesity and for evaluating efficacy of anti-obesity drugs.
2. Description of the Background Art
Obesity has been declared a public health hazard by the National Institutes of Health. To combat this health problem, both prophylactic and therapeutic approaches are necessary. For prophylactic purposes, it would be useful to be able to predict and measure a person's propensity or susceptibility to obesity For therapeutic purposes, a means for interfering with the development of adipocytes (fat cells) would be of great benefit. Furthermore, as a broader preventative approach to obesity, the ability to limit the fat content of food mammals would be of great importance. None of these desired objectives has been achieved.
Early-onset obesity cannot be efficiently controlled by a weight reduction program once the obesity is apparent. Therefore, a means for early detection of early-onset obesity is imperative for its prevention.
Proteins that are unique to adipocytes would serve as prime targets for approaches directed to prediction and control of obesity. There have been no reports of proteins that are preferentially synthesized in fat pads of obese animals as compared to normal controls. In fact the opposite has been found, wherein adipsin mRNA and protein is decreased in fat pads and in the circulation of obese animals compared to normal littermates (Flier, J. S. et al., Science 237:405-408 (1987).
It is known that differentiation of mammalian adipogenic cells, precursors of fat cells, into fully differentiated adipocytes, is accompanied by the induction of particular proteins. For example, glycerol-3-phosphate dehydrogenase (G3PDH) and fatty acid binding protein have similar levels of mRNA expression in obese and nonobese animals (Flier et al., supra). Lipolytic and lipogenic enzymes, which are absent in the undifferentiated cells, are also induced during adipocyte differentiation. A gene for human lipoprotein lipase has been cloned and shown to be homologous in sequence to the mouse lipoprotein lipase sequence (Wion, K. L. et al., Science 235: 1038-1041 (1987); Kirchgessner, T. G. et al., J.Biol. Chem. 262:8463-8466 (1987)). Structural similarity of the human to the rat lipoprotein lipase has also been disclosed (Holm, C. et al., Biochim. Biophys. Acta 1006:193-197 (1989)).
Several established cell lines which have the ability to undergo adipose differentiation in culture have been used as model systems to study adipose tissue development (Green, H. et al.. Cell 1:113-116 (1974); Negrel, R. et al., Proc. Natl. Acad. Sci. USA 75:6054-6058 (1978); Hiragun, A. et al., In Vitro 16:685-693 (1980); Serrero, G. et al., Anal. Biochem. 120:351-359 (1982); Harrison, J. J. et al., J. Cell Biol. 100:429-434 (1985); Chapman, A. B., et al., J. Biol. Chem. 259:15548-15555 (1984); Sparks, R. L. et al., Cancer Res. 46 5312-5319 (1986)). The differentiation of preadipocytes to adipocytes involve a series of events including morphological changes, induction of numerous proteins, alteration in hormonal receptors level and response (Green, H., In: Obesity: Cellular and Molecular Aspects (Ailhaud, A., ed.), Colloques de Inserm, pp 15-24 (1979); Coleman, R. A. et al. J. Biol. Chem. 253:7256-7261 (1978); Rubin, C. S. et al.. J. Biol. Chem. 252:3554-3557 (1977); Reed, B. C. et al. Proc. Natl. Acad. Sci. USA 74:4876-4880 (1977); Rubin, C. S. et al.. J. Biol. Chem. 253:7570-7578 (1978); Spiegelman, B. M. et al., J. Biol. Chem. 255:8811-8818 (1980)).
In the past several years, cDNA libraries of differentiated adipocytes have been constructed and screened either by differential hybridization (Chapman, A. B., et al., supra; Spiegelman, B. M. et al., J. Biol. Chem. 258:10083-10089 (1983); Bernlohr, D. A. et al., Proc. Natl. Acad. Sci. USA 81:5468-5472 (1984); Dani, C. et al., J. Biol. Chem. 264:10119-10125 (1989)) or by subtraction method (Smith, P. J. et al.. J. Biol. Chem. 263:9402-9408 (1988)). The application of these techniques has led to the molecular characterization of cDNAs coding for proteins which are fat specific and which can play an important role in adipose tissue development.
The present inventor's laboratory has studied adipose differentiation using a C3H mouse teratoma-derived cell line called 1246 which has several interesting characteristics. The 1246 cell line is a bipotential cell line able to differentiate not only into adipocytes but also into muscle cells (Serrero, G., In: Mammalian Cell Culture: The Use of Serum Free. Hormone Supolemented Media (Mather, J. P., ed.), Plenum Press, New York pp. 53-75 (1984)). A defined medium supporting cell proliferation and differentiation has been described (Serrero, G., 1982, supra). In these conditions, the cells stringently require insulin for both processes. The differentiation of 1246 cells is stimulated by growth hormone and inhibited by tumor necrosis factor-.alpha. (TNF.alpha.), transforming growth factor-.beta. (TGFB), epidermal growth factor and transforming growth factor-.alpha. (Serrero, G., In: Cellular Endocrinology: Hormonal Control of Embryonic and Cellular Differentiation (Serrero, G. et al., eds.,) Alan R. Liss, Inc., New York, pp. 191-204 (1986)).
Several differentiation-deficient cell lines which display increased tumorigenic properties were isolated from 1246 cells maintained in the absence of insulin (Serrero, G. In Vitro Cell. Devel. Biol. 21:537-540 (1985)). These cells were shown to produce in their conditioned media several polypeptide growth factors which either stimulate their proliferation or modulate negatively their ability to differentiate (Yamada, Y. et al., Proc. Natl. Acad. Sci. USA 85:5936-5940 (1988); Yamada, Y. et al., J. Cell. Physiol. 140:254-263 (1989)). Thus 1246 and its differentiation-deficient counterparts are a useful model system for investigating the molecular mechanisms of gene expression during adipose differentiation.